Antibacterial Activityand Comparison of the Volatile Oils of Tanacetum tenuisectum (Boiss.) Podl. Obtained by Three Different Methods of Extraction.

The essential oils obtained by hydrodistillation (HD), steam distillation (SD) and solvent free microwave extraction (SFME) from the stems and flowers of Tanacetumtenuisectum (Boiss.) Podl., which is endemic to Iran, were analyzed by combination of GC and GC/MS. Camphor (26.91 %, 27.23% and 25.52%), borneol (12.61%,11.48% and 7.62%) and 1,8-cineole (7.93%, 13.23% and 11.26%) were the main constituents of the HD,SD and SFME oils of Tanacetumtenuisectum respectively. All three oils were rich in regard to monoterpenes and small percentage of sesquiterpenes and non terpenoid compounds. Antibacterial activity of the essential oil of the plant was determined against six Gram positive and Gram negative bacteria. The results showed that this oil was active against all of the tested bacteria.


Introduction
The genus Tanacetum, which is an important member of the Compositaefamily, is widespread in Europe and Western Asia and consists about 150-200 species.The flora of Iran comprises 26 species of Tanacetumofwhich 12 are endemic (1,2). Some members of this genus have traditionally been used as a spicy additive for food, in cosmetics and as herbal remedies due to their biologically active compounds (3). Especially Tanacetumpartheniumhasbeenused sinceancienttimes for a variety of medicine proposes, and recently has gained considerableprominence due to its ability of alleviating the symptoms of migraine, athritisand psoriasis, and to inhibition of blood platelet aggregation (4).
This genus is also found to contain sesquiterpene lactones (16, 17) a large group of molecules with several biological activities (18,21).
As part of our ongoing work on the chemical analysis of volatile obtained from wild plants of Iran, we report the composition of the volatile oils of Tanacetumtenuisectum obtained by hydrodistillation, steam distillation and solvent free microwave extraction with its antibacterial activity.
Voucher specimens have been deposited at the Herbarium of the Research Institute of Forests and Rangelands (TARI), Tehran, Iran.

Isolation of the essential oils Distillation
Air-dried ground stems and flowers of the plant were separately subjected tohydrodistillation and steam distillation using a Clevenger type apparatus for 3h. The obtained essential oils were dried over anhydrous sodium sulfate and after filtration, stored at 4ºC until tested and analyzed. The yield was found to be 0.2% and 0.3% (w/w), respectively. Solvent Free microwave extraction SFME extractionwas performed in a Milestone ETHOS 1600 batch reactor, which is a multimode microwave reactor operating at 2455 MHZ with a maximum delivered power of 1000 W, variable in 10 W increments. The dimensions of the PTFE-coated cavity are 35×35×35 cm. During the experiment time, temperature, pressure, and power were controlled using the « easy-WAVE » software package. Temperature was monitored with the aid of a shielded thermocouple (ATC-300) inserted directly in to the sample container.
Ina typical SFMEprocedure, 250 g of dry stems and flowers of T.tenuisectum were moistened prior to extraction by soaking in water for 1 h, then draining off the excess water.This step is essential to give thestems and flowers the initial moisture. Moistened stems and flowers were next placed in a reactor without any added solvent or water. The essential oil is collected, dried with anhydrous sodium sulfate and stored at 0ºC until used.
Gas chromatography GC analysis was performed on Schimadzu15 A gas chromatograph equipped with a split/ splitless injector (25ºC) and a flame ionization detector (250ºC). Nitrogen was used as carrier gas (1 mL/min) and the capillary column used was DB-5 (50m0.2×mm, film thickness 0.32µm).The column temperature was kept at 60ºC for 3 min and then heated to 220ºC witha 5ºC/ min rate and kept constant at 220ºC for 5 min. Relative percentage amount were calculated from peakarea usinga Schimadzu C-R4 Achromatopac without the use of correction factors.

Gas chromatography -mass spectrometry
Analysis was performed using a Hewlett-Packard 5973 with a HP-5MS column (30m0.25× mm, film thickness 0.25 µm). The column temperature waskept at 60ºC for 3 min and programmed to 220ºC at a rate of 5ºC/min and kept constant at 220ºC/min for 5 min.The flow rate of Helium as carrier gas with (1 mL/ min).
Mass spectrometry wastaken at 70 eV. The retention indices for all the components were determined according to the Van Den Door method, using n-alkanes as standards (33).
The compounds were identified by (RRI, DB5) with those reported in the literature and by comparison of their mass spectra with the Wiley library or with the published mass spectra (34, 35).

Antibacterial assay
The antibacterial activity of the essential oil from the aerial parts of Tanacetumtenuisectum was evaluated by disc diffusion method using Mueller-Hinton Agar (36). The antibacterial activity of the essential oil of the plant was tested against three Gram-positive and three Gramnegative bacteria.
The Gram-positive bacteria included Staphylococcus aureusATCC 25923, A serial dilution of the oil was prepared in Mueller-Hinton Broth forbacteria. The oil was diluted by the water and ethanol solvents. The solvents, at an appropriate concentration were also used as a negative centrol. The standardized suspension of bacteria was incubated in to each tube. The tubes were incubated at37ºCfor 24h. Thelowestoil concentration, where there was no visible growth, was the Minimum Inhibitory Concentration (MIC) when compared to control.
To determine the Minimum Bactericidal Concentration (MBC), for each set of test tubes in the MIC determination, a loopful of broth was collected from those tubes which did not show any growth and incubated on Muller-Hinton Agar by streaking. Plates incubated with bacterial were then incubated at 37ºC for 24 h. After incubation, the concentration at which no visible growth was seen was noted as MBC for bacteria. All the experiments were carried out in triplicate and mean calculated.

Results and Discussion
The identified volatile components and their peak area percentages of the stems and flowers of Tanacetumtenuisectumobtained by hydrodistilliation, steam distillation and solvent free microwave extraction are given in Table  1. The components are listed in order of their elution on the DB-5 column.
As it is shown from the Table 1, about 96.87% (34 components) of the hydrodistilled oil, 91.32% (46 constituents) of the steam distilled oil and 95.1% (44 components) of the solvent free microwave extraction oil of T.tenuisectumwere identified.
Accordingto these results, the composition of the three oils show significant similarity for the concentration of the main components. All three oils were rich in regard tomonoterpenes (55.14%, 68.69%and 55.97%, respectively), while the sesquiterpenes fraction was (27.27%, 13.89% and 24.47%, respectively). The nonterpenoid fraction was relatively small, representing 14.46%, 8.74%, and 14.66%, respectively.
The oils obtained by hydrodistillation of the leaves and flowers of T.dumosum growing wild in Iran were investigated. The main constituents Table 1. Comparative chemical composition (%) of Tanacetumtenuisectum oil obtained by HD, SD and SFME. Note: a Compounds listed in order of elution from HP-5 MS column; b Retention indices to C 8 -C 24 n-alkanes on HP-5 MS column; t= trace ( < 0.1% ).  (31).
The dominant compound in the flower and stem oils ofT. chiliophyllum, from Turkey, was camphor (17.3% and 10.4%) respectively,while root oil of the plant was characterized with hexadecanoic acid (37.5 %) (37).
Baser et al. reported the oils from the flowers of T. zahlbruckneri and flowers and stems of T. tabrisianum from Turkey.
Results of the antibacterial activity of the essential oil of Tanacetumtenuisectum  (42,43).The antibacterial effects of borneol were also reported (44). As aresult of these findings, antibacterial activity of T. tenuisectum oil could be attributed to 1,8 cineol, camphor and borneol. The present study confirms that there is a positive correlation between the chemical content of the oils and their antibacterial activities.